This invention relates to nucleozymes, mixed nucleic acid polymers having catalytic activity. The invention also relates to methods of preparing and using nucleozymes.
Proteins were the only known catalysts of cellular reactions until the discovery of RNA catalysts (ribozymes). In some instances, the folded structure of a ribozyme catalyses a cleavage reaction on another part of the same molecule (cis-reaction). In other instances (trans-reaction), the ribozyme may also act as a catalyst on another RNA or DNA molecule (substrate) by cleaving or ligating pieces of the substrate without changing itself in the process. (Zaug et al. Science, Vol. 231, pp. 470-75, 1986; Cech Science, Vol. 236, pp. 1532-39, 1987).
A well-characterized example of a ribozyme is the self-splicing Group I intron from the nuclear rRNA of Tetrahymena thermophila. An intron is an intervening sequence in a eukaryotic gene which does not encode a protein or in rare cases encodes a different protein. Introns are transcribed along with coding sequences (exons) to produce precursor RNA. The introns are removed from the precursor RNA and the exons are ligated by RNA cleaving and splicing steps. The Group I intron or ribozyme of T. thermophila catalyzes its own removal from the precursor RNA molecule. (Kruger et al. Cell 31:147-157, (1982); Zaug et al. (1986)). The self-splicing ribozyme catalyzes a variety of phosphodiester transfer reactions. The ribozyme can act as a ribonuclease, ligase, phosphotransferase, acid phosphatase, polymerase and RNA restriction endonuclease (Zaug, A. J., et al., Science 231:470-475 (1986); Zaug, A. J., et al., Nature 324:429-433 (1986); Zaug, A. J., et al., Biochemistry 25:4478-4482 (1986); Been, M. D., et al., Science 239:1412-1416 (1988); Doudna et al., Nature 339:519-522 (1989); all incorporated by reference herein).
The xe2x80x9chammerheadxe2x80x9d and xe2x80x9chairpinxe2x80x9d ribozymes also have been studied and described (Perreault et al., Nature 344:565-567 (1990); Perreault et al. Biochemistry 30:4020-25 (1991); Yang et al. Biochemistry 29:11156-60 (1990); Chowrira et al. Biochemistry 30:8518-22 (1991); Uhlenbeck Nature, 328:596-600 (1987)). The hammerhead ribozyme forms a stem loop secondary structure to form the catalytically active molecule. The hairpin ribozyme has a structure resembling a hairpin.
Although ribozymes are intriguing molecules, their use for in vivo applications is limited if not precluded. The all-RNA molecules are susceptible to degradation from enzymes (RNAses) present in vivo. There presently is no way known to inventors for delivering such molecules to the intended site in an active form.
The present invention is based on the discovery that ribozymes have catalytically critical sites and that it is not necessary to have an all-RNA molecule to have catalytic activity. The ribozyme like molecules of the invention or xe2x80x9cnucleozymesxe2x80x9d have ribonucleotides or nucleic acid analogues (hereinafter NAAs) at catalytically critical sites and NAAs or deoxyribonucleotides at non-catalytically critical sites. The preferred nucleozymes have ribonucleotides at catalytically critical sites. Nucleozymes have catalytic activity on the same substrates as their ribozyme counterparts.
The nucleozymes of the present invention thus essentially are modified ribozymes having at least a portion, or all, of the ribonucleotides replaced with deoxyribonucleotides or NAAs. The nucleozymes are significantly more resistant to degradation than their all-RNA ribozyme counterparts because the chemicals or enzymes present in vivo do not recognize the nucleic acid internucleotide bonds. The resistance can be to either enzymatic or chemical degradation. Preferably, a majority of the ribonucleotides of the ribozyme are replaced with deoxyribonucleotides or NAAs. The stability of the nucleozymes allows them to be useful as therapeutic agents whereas ribozymes would be cleaved and rendered inactive by enzymes, e.g. RNAses, present in vivo.
The nucleozymes of the present invention are chimeric nucleic acid polymers having catalytic activity due to and preferably optimized by the presence of RNA or a NAA at a catalytically critical site. The present invention provides chemistry which allows synthesis of the chimeric polymers and the determination of catalytically critical sites. The sites may be determined by varying the location of deoxyribonucleotides in a chimeric polymer and determining the locations responsible for or related to the chimeric polymer""s ability to catalyze.
The present invention also pertains to a method for making a chimeric polymer. The polymers are made by phosphitylating protected ribonucleotides or NAAs units under conditions to form substantially pure-protected phosphoramidites or synthons of a single isomer. The protected phosphoramidites are coupled to each other forming a protected chimeric nucleic acid chain. The protecting groups are removed from the chimeric nucleic acid chain under conditions which completely deprotect the polymer.
The method of the present invention alleviates problems associated with a known prior art method for preparing chimeric RNA/DNA polymers (Perreault et al. Nature 344:565-567 (1990); Wu et al. Journal of the American Chemical Society 111:8531-33 (1989)). The prior art synthetic method for making chimeric polymers had problems with the migration of the protecting groups during the phosphitylating step, difficulty in removing the protecting groups and also has problems resulting from the process of removing the protecting groups in the deprotection step. The former problem results in the production of monomer units having protecting groups in an undesired position. The latter problem results, in many cases, in a) nucleotide modification, b) phosphodiester linkage isomerization, and c) to retention of a substantial amount of protecting groups on the polymer resulting in a non-functional polymer. The first problem was overcome in the present invention by selecting a catalyst capable of minimizing migration of protecting groups, e.g., a combination of 2,4,6-collidine and N-methylimidazole. The problem in removing the protecting groups was eliminated by deprotecting the mixed polymer in the presence of ethanolic ammonia.
The present invention also pertains to methods of using the nucleozymes. The nucleozymes may be used to perform the same catalytic functions as their all-RNA ribozyme counterparts. For example, a nucleozyme may be used as a ribonuclease, ligase, phosphotransferase, acid phosphatase, polymerase, or an RNA restriction endonuclease. The nucleozymes may be used to selectively cleave and ligate substrates by contacting the substrates with a nucleozyme such that the nucleozyme targets a specific sequence in the substrate for cleavage or ligation. The nucleozymes may be used as polymerases to polymerize the production of an oligoribonucleotide or an oligodeoxyribonucleotide. The nucleozymes also may be used in place of antisense RNA technology.
The nucleozymes also may be used as therapeutic agents introduced in vivo due to their resistance to chemical and enzymatic degradation. The nucleozymes may be used, for example, in a method for treating a subject for a retrovirus associated disease, e.g., human immunodeficiency virus (HIV). The method involves administering a therapeutically effective amount of at least one nucleozyme to the subject such that the nucleozyme cleaves the RNA genome of the retrovirus rendering it inactive. A plurality of nucleozymes also may be administered if it is desirable to target more than one sequence in the RNA genome.
A nucleozyme may be provided in a pharmaceutical composition. The pharmaceutical composition would include at least one nucleozyme and a pharmaceutically acceptable carrier.
It is an object of the present invention to provide a nucleozyme capable of maintaining its catalytic properties in vivo.
It is an object of the present invention to provide a chimeric nucleic acid polymer having catalytic activity.
It is yet another object of the present invention to provide a method for preparing chimeric polymers which are free of protecting groups and undesired isomeric side products.
It is yet another object of the present invention to provide a homogenous chimeric polymer.